Abstract

The Nrf2 transcription factor belongs to the Cap'n'collar family, named after the founding member of this group, the product of the Drosophila Cap'n'collar gene. The encoded protein, Cap'n'collar, abbreviated Cnc, offers a convenient and accessible model to study the structure, function, and biology of Nrf2 transcription factors at the organismic, tissular, cellular, and molecular levels, using the powerful genetic, genomic, and biochemical tools available in Drosophila. In this review we provide an account of the original identification of Cnc as a regulator of embryonic development. We then describe the discovery of Nrf2-like functions of Cnc and its role in acute stress signaling and aging. The establishment of Drosophila as a model organism in which the mechanisms and functions of Nrf2 signaling can be studied has led to several discoveries: the regulation of stem cell activity by an Nrf2-mediated redox mechanism, the interaction of Nrf2 with p62 and Myc in the control of tissue growth and the unfolded protein response, and more. Several of these more recent lines of investigation are highlighted. Model organisms such as the fly and the worm remain powerful experimental platforms that can help to unravel the many remaining puzzles regarding the role of Nrf2 and its relatives in controlling the physiology and maintaining the health of multicellular organisms.

The cartoon depicts the stages of Drosophila development (embryogenesis, pink; larval and pupal stages, green) and adulthood (blue). Females deposit fertilized eggs. Upon completion of embryogenesis the first larval stage (1. instar) hatches. Subsequent molting gives rise to second and third instar larvae. At the end of the last larval stage pupae are formed and metamorphosis to the adult form (imago) commences. The durations of these different developmental phases under optimal growth conditions are indicated. Cnc-regulated biological processes that occur at different stages of the Drosophila life cycle and are discussed in this review are listed.

A representation of cap’n’collar transcription factors found in humans and Drosophila

Structural domains were identified using MAFFT and T-COFFEE to compare publicly available amino acid sequences of human Nrf1, Nrf2, Nrf3, and different protein isoforms expressed from the Drosophila cnc locus (). The N-terminal domains (NTD) present in Nrf1, Nrf3, CncC, and CncO contain the NHB1/2 subdomains that are believed to play an important role in targeting these proteins to the endoplasmic reticulum (). The NEH2 domains contain the DIDLID/DLG and the ETGE motifs that are essential for Keap1 binding as well as Keap1-mediated degradation of these proteins (; ). NEH4 and NEH5 are transactivation domains involved in the recruitment of the CREB binding protein, CBP (). The SR region is a serine-rich region of the Nrf1 protein. The highly conserved cap’n’collar (CNC) motif as well as the basic region and leucine zipper, all of which play a key role in DNA binding, are located within the NEH1 domain (). The NEH3 domain is thought to be involved in transactivation by recruitment of CHD6, a chromo-ATPase/helicase DNA binding protein (). The NEH6 domain contains a putative repression site for β-TrCP and several conserved serine residues that may be phosphorylated by GSK-3 ().

Amino acid sequence homology between Cnc, Keap1 and small Maf proteins of human and Drosophila

A) Alignment of key functional elements in Nrf1, Nrf2, and CncC. Multisequence alignment using MAFFT, T-COFFEE, and BOXSHADE were used to compare publicly available amino acid sequences of human Nrf1, Nrf2, and CncC (). Residues that are conserved between at least two of the sequences are shaded in black and residues with similar characteristics are shaded in gray. Domains and features described in are highlighted.B) Alignment of Human and Drosophila Keap1. Multisequence alignment using MAFFT, T-COFFEE, and BOXSHADE were used to compare publicly available amino acid sequences of human Keap1 (hKeap1) and Drosophila Keap1 (dKeap1) (). Residues that are conserved are shaded in black and residues with similar characteristics are shaded in gray. The BTB (Broad complex, Tramtrack, and Bric-a-Brac) domain contains key cysteine residues important for sensing oxidative stress (). The linker domain plays a role in targeting Nrf2 for ubiquitination leading to its degradation as well as maintaining localization of the Nrf2-Keap1 complex in the cytoplasm (). The Kelch repeat domain is responsible for Nrf2 interaction by interacting with the NEH2 regulatory domain of Nrf2 ().C) Alignment of Mouse and Drosophila small Maf proteins. Multisequence alignment using MAFFT, T-COFFEE, and BOXSHADE were used to compare publicly available amino acid sequences of human Keap1 and Drosophila Keap1 (). Residues that are conserved are shaded in black and residues with similar characteristics are shaded in gray. The basic-leucine zipper region is highlighted.

As in mammals, Nrf2 activity can be activated in Drosophila in response to a variety of different types of cells stress. Flies carrying a CncC responsive ARE-GFP reporter and, for comparison, a separate AP-1 (Fos & Jun) responsive TRE-GFP GFP reporter () were exposed to dietary Paraquat, H2O2, DEM, and sodium aresenite. Controls treated with matching solvents and photographed under the same exposure are shown. A mutated, Nrf2 unresponsive reporter (mRE-RFP) is not affected by these treatment conditions.